This invention relates to an improved method and apparatus for testing samples for the presence of microorganisms, such as Salmonella and Listeria, and Campylobacter.
Salmonella Bacterium.
Salmonella is a genus of bacteria responsible for a wide range of quite serious enteric infections often spread through food, water, and feeds. Salmonellosis usually is the primary bacterial cause of common foodborne diseases in North American and Europe according to national public health statistics. The disease can range from a self-limiting gastroenteritis with mild gastroenteric symptoms of diarrhea, fever, vomiting, and/or cramping to very severe, life-threatening gastroenteritis with or without bacteriemia. Although salmonellosis is generally considered a self-limiting illness, estimates of 9,000 deaths per year from approximately 2,000,000 cases annually have been reported for the United States. However, although the health statistics are useful in detecting trends or major outbreaks, the numbers reported are significant underestimates of actual cases. It is generally believed that only 1-10% of the cases are reported to public health authorities. Furthermore, due to changes in eating habits and international trade of ingredients and finished product, the incidence of salmonellosis in the United States and many other countries is on the increase. Cost estimates of almost $30,000 per case per outbreak resulting from investigated Salmonella outbreaks have been made. Thus, the detection of Salmonella, as well as other food-borne pathogens, in foods, feeds, and the environment is of great public health concern.
Listeria Bacterium.
Interest in the occurrence of Listeria and particularly Listeria monocytogenes in food and food-processing environments continues unabated as the result of four listeriosis outbreaks during the 1980's that were linked to coleslaw, pasteurized milk and two types of cheese--California-made, Jalisco-brand Mexican-style cheese and Swiss-made, Vacherin Mont d'Or soft-ripened cheese. Overall, these outbreaks resulted in over 500 cases of listeriosis and included at least 110 deaths.
In response to these outbreaks, the United States and many European countries began large-scale programs to determine the incidence of this pathogen in a wide variety of foods. As of 1990, a total of 76 Class I recalls were issued in the United States for Listeria contaminated dairy, meat and seafood products which resulted in staggering financial losses for the food industry. A similar situation is known to exist in Europe with the isolation of L. monocytogenes from all food types examined in the United Kingdom except baked beans and bottled water. Nonetheless, European recalls of Listeria-contaminated products have been relatively rare.
Present evidence indicates that Listeria enters most foods as a post-processing contaminant. Hence, the key to producing a Listeria-free product is to eliminate this organism from the food-processing environment. This is also the main principle on which the widely recognized Hazard Analysis Critical Control Point (HACCP) concept was founded over 20 years ago. Given this information, a clear need exists to optimize recovery of Listeria from the food-processing environment.
Present Methods Of Detecting Microorganisms Such As Salmonella And Listeria
Microorganisms found in foods and ingredients are often stressed as a result of food processing conditions. This applies to microorganisms that are human pathogens like Salmonella and Listeria, or other pathogens, as well as food spoilage microbes. Many of the processes used in manufacture of foodstuffs are designed to reduce or eliminate the microbiological population in or on the food to prevent illness in the consumer or to extend shelf-life of the product. Unless the process is sterilization, some cells survive processing. The cells which survive are usually injured and debilitated by processing and are consequently more difficult to isolate. Salmonella and Listeria are examples of microorganisms affected in such a manner.
The problem is how to detect and isolate the debilitated, stressed Salmonella or Listeria which, if present, are present only in very low numbers and in the presence of other competing microorganisms. Furthermore, the inherent variety and variability of foods, ingredients, and feeds and environments sampled can affect the incidence of isolation.
Several methods have been used to obtain samples of liquids and foods or samples of environments in which foods are processed or packaged for Salmonella and Listeria testing. Solid food samples have been obtained by cutting pieces from food to be tested. The pieces are then ground for purposes of the testing that is discussed below. Samples of milk or other liquids have been obtained by use of aseptic valves, syringes, pipettes, cups, or direct from containers. Environmental samples have been obtained by swabbing a surface with sponges or other absorbent materials, such as gauze.
In addition to food testing, water samples have been tested for Salmonella and other microorganisms. Water has been collected for such testing in containers, and also by absorbent materials such as tampons, as is discussed in Venkateswaran, Takai, Navarro, Nakano, Hashimoto, and Siebeling, Ecology of Vibrio cholerae Non-01 and Salmonella spp. and Role of Zooplankton in Their Seasonal Distribution in Fukuyama Coastal Waters, Japan, Applied and Environmental Microbiology, pp. 1591-1598 June 1989). That article noted that, better isolation of an allochthonous pathogen, viz., Salmonella spp., was noticed from the water samples when swabs were employed. Another paper discloses the use of a water sampling device consisting of a wire:gauze:wire sandwich. (Venkateswaran et al., 1989 Applied and Environmental Microbiology 556:1591-1598.) Although the article noted that there appeared to be better isolation of Salmonella from water samples if such swabs were employed, no information or data was given in the publication, and no attempt was made to explain that phenomena.
Current Methods of Salmonella Detection
The Bacteriological Analytical Manual, (BAM) published by the Food and Drug Administration (FDA), Ch. 10 (6th Ed. 1984) ISBN 0-935584-29-3), and the Official Methods of Analysis, published by the Association of Official Analytical Chemists, pp. 467-476 (15th Ed. 1990) (AOAC) describe the current methodology for detection, isolation, and identification of Salmonella from samples of the type discussed above. In general terms the five steps are: 1) Pre-enrichment, 2) Selective enrichment, 3) Selective plating, 4) Biochemical screening, and 5) Serotyping.
The first step, pre-enrichment, consists of introducing the sample into a non-selective, nutritious, fluid medium at a 1:10 sample:medium ratio to allow the debilitated, stressed Salmonella cells, if present, to recuperate to a stable physiological state. Depending on the product, this pre-enrichment medium can differ. However, if a highly contaminated product, such as a raw flesh product, is being analyzed, then the testing is initiated with the selective enrichments.
After incubation for 24 hours at 35.degree. C., a 1 milliliter (mL) quantity of the pre-enrichment culture is transferred to selective enrichment. Selective enrichment uses two specifically designed liquid media known as tetrathionate broth and selenite cysteine broth, to allow continued proliferation of Salmonella while restricting the growth of most other competitor bacteria through use of selectively inhibitory reagents. After a second incubation of 24 hours at 35.degree. C., the selective enrichments are streak plated onto three solid selective agar media. These media are known as xylose lysine desoxycholate (XLD), Hektoen enteric (HE), and bismuth sulphite (BS) agars. The selective plating media restrict growth of non-Salmonella microorganisms while allowing visual recognition of pure, discrete suspect-Salmonella colonies. The Salmonella-suspect colonies are picked for biochemical screening to eliminate other microorganisms with similar identification characteristics as Salmonella. The specific metabolic characteristics of most Salmonella allow a differentiation from other microorganisms and allows a tentative generic identification as Salmonella. Serotyping provides the species-specific identification of the tentatively identified Salmonella isolate.
The exact description of the conventional materials and procedures is described by the FDA in BAM, the AOAC, and the Compendium of Methods for the Microbioloqical Examination of Foods, pages 286-326 (2nd Ed., American Public Health Association 1984).
Current Methods of Listeria Detection
The present procedure for recovering Listeria from food-processing environments is based on the current USDA method (FIG. 6) with the sample collected on a sterile sponge. In this procedure, an ethylene oxide-sterilized cellulose sponge is removed from its protective package using surgical gloves and placed in a sterile Whirl-Pak bag. After rehydrating the dry sponge with 50 mL of neutralizing buffer, the wet sponge is removed from the bag using a surgical glove and the sample is collected by wiping the sponge over the desired surface i.e. floor, drain, pipe or a piece of equipment. The sponge is then returned to the same Whirl-Pak bag which is appropriately labeled and shipped to the laboratory by overnight delivery for analysis. Upon receipt of the sample, ca. 50 mL of UVM Broth a selective enrichment broth for Listeria is added to the bag containing the sponge. After squeezing the bag to incorporate the contents of the sponge in the broth, the sample is incubated at 30.degree. C. for 24 h. Following incubation, 0.1 mL of the broth is pipetted into an appropriately labeled test tube containing 10 mL of Fraser Broth, a semi-selective differential enrichment broth that turns black in the presence of metabolically active Listeria spp. and a few other bacteria. After 24 and 48 h of incubation at 35.degree. C., the Fraser Broth enrichment is streaked to appropriately labeled Petri plates containing Lithium Chloride-Phenylethanol-Moxalactam Agar (LPM) and Modified Oxford Agar (MOX), both agars of which will selectively differentiate Listeria from other bacteria. Following 24 and 48 h of incubation at 35.degree. C., five well isolated Listeria-like colonies from each plate are streaked to Trypticase Soy Agar+Yeast Extract (TSAYE), a nutritious non-selective medium for re-isolation and purification of the suspected Listeria isolate. After 24 h of incubation at 35.degree. C., each organism is examined for catalase activity. Gram-positive, catalase-positive, bacilli are then further characterized using the CAMP reaction, biochemical and serological reactions for identification of Listeria spp., including L. monocytogenes.
Current Methods Of Detection Of Other Microorganisms
The exact description of the conventional materials and procedures for testing other microorganisms is described by the FDA in BAM, the AOAC, and the Compendium of Methods for the Microbiological Examination of Foods, (2nd Ed., American Public Health Association 1984), and is known to those skilled in the art. Each of the methods of isolation involves the steps of transferring a sample into one or more enrichment media, and then transferring a sample from the enrichment medium into another medium for isolation. For example, the methods of detection of E. coli and other coliforms involves placing a food sample homogenate in an lauryl sulfate tryptose (LST) medium, incubating the sample in the broth, transferring a sample to a brilliant green lactose bile broth, and performing a biochemical analysis on positive samples thereafter. According to BAM, isolation of Shigella involves transferring a sample into a broth, incubating the broth, and then removing a portion of the incubated broth and inoculating MacConkey agar, and streaking it on the agar. Similar transfers are involved with the detection of other microorganisms, such as Campylobacter, Vibrio species, Staphylococcus aureus, Bacillus cereus, and Clostridium botulinum.
There are several drawbacks to the present methods for detection, isolation, and identification of microorganisms, such as Salmonella and Listeria. First, the methods currently used to isolate those and other microorganisms require separate steps of transferring samples. For example, in the case of Salmonella and Listeria, after the sample is initially incubated, a portion of it must be transferred to a pre-enrichment broth; and second, after further incubation, samples of selective enrichment must be streak plated onto agar media. The first transfer involves the use of a pipette or a syringe. The second transfer also involves the use of a separate device. In both cases, workers may be exposed to the bacteria and the sample may be subject to contamination. In addition, there is a danger of mislabeling samples. The same problems exist with the methods used to detect other microorganisms.